CN116271032A - Drug delivery system for enhancing tumor immunotherapy and preparation and application thereof - Google Patents

Abstract

The invention discloses a drug delivery system for enhancing tumor immunotherapy and preparation and application thereof, belonging to the field of biological medicine. The drug delivery system for enhancing tumor immunotherapy is platelet exosome hybrid liposome (DCHL) which carries AIE photosensitizer and Chloroperoxidase (CPO). The drug delivery system for enhancing tumor immunotherapy of the invention loads and delivers CPO and a novel AIE photosensitizer DPDPDPDPy to a tumor site, and can realize continuous singlet oxygen generation and enhance EDT and tumor immunotherapy. The invention provides a new scheme for tumor treatment.

Description

Drug delivery system for enhancing tumor immunotherapy and preparation and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a drug delivery system for enhancing tumor immunotherapy, and preparation and application thereof.

Background

Immunotherapy has become the fourth largest treatment for tumors following surgery, radiation therapy and chemotherapy, and is one of the most advanced research fields of applied research and clinical medical practice in recent years. Moreover, with the rapid advancement of research on the mechanism of immunotherapy, it has been successfully applied to clinical treatment of cancer. Active oxygen-based anti-tumor therapies (e.g., photodynamic therapy, radiotherapy, chemo-dynamic therapy, sonodynamic therapy, etc.) can effectively cause Immunogenic Cell Death (ICD) of tumor cells and T-cell infiltration, resulting in systemic immune responses, which have been confirmed by a number of previous studies and constitute a very effective approach to promote tumor immune efficacy. Among them, photosensitizers (AIEgens) having aggregation-induced emission effect, which can effectively generate Reactive Oxygen Species (ROS) under light conditions to kill tumor cells, have been developed for the treatment of cancer. In addition, AIEgens have the advantages of simple preparation, photo-bleaching resistance, excellent fluorescence property, good biocompatibility and the like, are widely applied to the field of photodynamic therapy (PDT), have made great progress in recent years, and have potential medical application values. However, ROS are generally short in lifetime (< 0.04 μs), and after the end of the photoreaction, ROS annihilate rapidly, so their range is relatively limited. Continuous illumination is required for high ROS production, but long-term illumination can cause skin damage and other adverse side effects. Thus, there is a need to develop new strategies that can allow sustained generation of ROS at the tumor site to promote the efficacy of PDT and immunotherapy.

Enzyme Dynamic Therapy (EDT) is a potential candidate for promoting tumor immunotherapy, and Chloroperoxidase (CPO) can catalyze the reaction of chloride with hydrogen peroxide (H ₂ O ₂ ) Hypochlorous acid (HClO) is generated to form singlet oxygen ¹ O2). EDT processes are limited by the hydrogen peroxide content, which is limited in cells and the overexpression of glutathione inhibits the anti-tumor effect of EDT, thus requiring modification of tumor microenvironment to enhance the effect of EDT.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and providing a drug delivery system for enhancing tumor immunotherapy and preparation and application thereof.

The aim of the invention is achieved by the following technical scheme:

an administration system for enhancing tumor immunotherapy is a platelet exosome hybrid liposome which is loaded with AIE photosensitizer and chloroperoxidase together and is named DCHL.

In some embodiments, the AIE photosensitizer is DPDPDPY with a structure shown in the following formula, and the DPDPDPY has higher singlet oxygen generating capacity and photobleaching resistance.

DPDPy is preferably prepared by a process comprising the steps of: dissolving 4-dimethylamino cinnamaldehyde and 1, 4-dimethyl pyridine iodide in a solvent, adding piperidine, and carrying out reflux reaction to obtain DPDPDPy. Wherein the solvent is preferably ethanol.

The preparation method of the drug delivery system for enhancing tumor immunotherapy is schematically shown in fig. 1, and comprises the following steps:

(1) Dissolving 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), cholesterol (Cho) and AIE photosensitizer in an organic solvent, and forming a film by spin evaporation; and (3) adding a solution containing Chloroperoxidase (CPO) to hydrate the film, and performing ultrasonic treatment to obtain the liposome carrying the AIE photosensitizer and the chloroperoxidase.

(2) Mixing the AIE photosensitizer and chloroperoxidase-loaded liposome and platelet exosome, and extruding to obtain AIE photosensitizer and chloroperoxidase-loaded platelet exosome hybrid liposome (DCHL).

In the preparation method of the drug delivery system for enhancing tumor immunotherapy, the organic solvent is preferably chloroform; the rotary steaming is preferably carried out at 30-70 ℃; the hydration is preferably carried out at 37 ℃; the solution containing the chloroperoxidase is preferably PBS solution containing the chloroperoxidase, and the extrusion is preferably performed by using a polycarbonate film with a pore diameter of 100 nm.

The drug delivery system for enhancing tumor immunotherapy is applied to the preparation of antitumor drugs.

An antitumor drug comprises the drug delivery system for enhancing tumor immunotherapy, and can further comprise a pharmaceutically acceptable carrier or excipient.

The action principle of the drug delivery system (DCHL) for enhancing tumor immunotherapy is shown in figure 1, after DCHL targets tumor cells, the liposome is destroyed under illumination, DPDPDPy and CPO are released, and the DPDPy generates singlet oxygen, thereby causing mitochondrial damage and massive hydrogen peroxide generation. The intracellular chloride and hydrogen peroxide can then further generate hypochlorous acid and singlet oxygen under the action of CPO, which can continue to cause oxidative stress and immunogenic death of the tumor. Finally, tumor-associated antigens can stimulate the immune system, causing Dendritic Cell (DC) maturation and T cell activation and infiltration, leading to a systemic immune response.

The invention has the advantages and beneficial effects that: the continuous generation of active oxygen plays a key role in promoting tumor photo-immunotherapy, and the administration system for enhancing tumor immunotherapy of the invention loads and delivers CPO and a novel AIE photosensitizer DPDPDPDPy to a tumor site, so that continuous singlet oxygen generation and enhancement of EDT and tumor immunotherapy can be realized. The invention provides a new scheme for tumor treatment.

Drawings

FIG. 1 is a schematic representation of the preparation of hybrid platelet exosomes liposomes co-loaded with AIE photosensitizer DPDPDPy and Chloroperoxidase (CPO) and their use for uninterrupted singlet oxygen generation and enhanced tumor immunotherapy.

FIG. 2 is a characterization of the AIE photosensitizer DPDPDPDpy. (A) PL spectra of DPDPy in toluene/water containing different concentrations of toluene. (B) D (D)Plot of AIE values of PDPy at 700nm versus different toluene concentrations in toluene/water mixtures. AIE refers to the reaction of the toluene moiety (f _t ) And 0%f _t Ratio of PL intensities. (C) Decomposition Rate of DPBF in the Presence or absence of allergen and light irradiation, wherein A ₀ And A is the absorbance of DPBF at 420nm before and after irradiation, respectively. (D) White light irradiation (0.1W/cm) ² ) Absorption spectra of DPDPDPDPy before and after 5min.

FIG. 3 is a characterization of platelet exosome hybrid liposomes (DCHL) co-loaded with DPDPY and CPO. (a) TEM images of DCL, PEV and DCHL. (B) diameter distribution of DCHL. (C) Hydrodynamic diameters and zeta potentials of different formulations suspended in PBS. Data are expressed as mean ± SD (n=3). (D) stability of DCHL in PBS or PBS containing 10% FBS. Data are expressed as mean ± SD (n=3). (E) PEV membranes labeled with fluorochromes (Dil and DiD) were fused to more and more Liposomes (LIP), and the recovery of fluorescence from the donor (Dil) was measured to evaluate the fusion. (F) PEV markers CD41 and P-selectin were detected using western blotting. (G) Absorption spectra of CPO (in PBS), DCHL (in PBS), blank PEV-mix liposomes (BL, in PBS), and DPDPy (1% DMSO component). (H) absorption spectra of DPBF treated with different formulations. (I) Absorption spectra of DPBF at different irradiation time points in the presence of DCHL. (J) cumulative release profile of CPO under different conditions. Data are expressed as mean ± SD (n=3). (K) CLSM images of cancer cells were incubated with DiO-labeled DCL or DCHL for 1 h. Blue: DAPI; red: diO. Scale bar: 10 μm. (L) DiO fluorescence intensity in FIG. 3K, PBS-treated cells served as control.

Fig. 4 is a graph showing that DCHL can achieve uninterrupted singlet oxygen production by tumor cells. (A) Fluorescent images of 4T1 cells treated with different formulations stained with mitochondrial red fluorescent Violet and DAPI. Scale bar: 10 μm. (B-C) 4T1 cell H after different treatments ₂ O ₂ And the corresponding fluorescence intensity (n=3). Scale bar: 10 μm. (D-E) differently treated 4T1 cells 0 and 4h after irradiation ¹ O2 fluorescence imaging, and corresponding flow cytometry fluorescence intensity analysis. Scale bar: 10 μm. * P:<0.005; student t test.

FIG. 5 is the growth inhibitory capacity and immunogenic death inducing effect of DCHL on 4T1 cells. (A) Cell viability of 4T1 cells after different treatments (n=3). (B) 4T1 cell viability (n=3) after treatment with different DPDPy concentrations dchl+l. (C) content of GSH in 4T1 cells treated differently (n=3). (D) HMGB1 released by 4T1 tumor cells after 24h of each treatment was quantitatively examined. (E) Immunofluorescent staining (Green: CRT, blue: DAPI) and fluorescence intensity of CRT exposure of various treated 4T1 tumor cells. Scale bar: 10 μm. * P <0.05, P <0.005; student t test.

Figure 6 is the circulating capacity and tumor targeting capacity of DCHL in tumor-bearing mice. (A) Pharmacokinetic profiles of DCL and DCHL in tumor-bearing mice. (B) In vitro images of tumor tissues and organs collected in 4T1 tumor-bearing mice 24h after DCL or DCHL injection.

FIG. 7 is the antitumor ability of DCHL in tumor-bearing mice. Schematic of 4T1 tumor treatment. (B-C) change in Primary (Primary) and Distant tumor (distance) volumes after different treatments in tumor-bearing mice. (D) tumor weights of different treated mice. (E) remote tumor growth curve for each mouse. (F) Treatment of induced lymph node dendritic cell maturation and CD4 in distant tumors ⁺ /CD8 ⁺ Flow cytometry analysis of T lymphocytes. (G) HE, SOSG and CRT staining analyses of primary tumor tissues of different treatments. Scale bar: HE and SOSG were 40 μm and CRT was 20. Mu.m. * P<0.05，***P<0.005; student t test.

FIG. 8 is the levels of the pro-inflammatory cytokines IFN-gamma (A) and TNF-alpha (B) in serum of different treated mice.

FIG. 9 is a graph showing CD8 in distant tumors of different treated mice ⁺ Number of T cells.

Fig. 10 is a view of the good biosafety of DCHL. (A) mice body weight during administration. (B) biochemical analysis of blood of mice after DCHL treatment. (C) Main organ HE staining of mice after DCHL treatment.

FIG. 11 is the effect of DCHL on 4T1 tumor re-excitation and recurrence. Schematic of (A) 4T1 tumor re-excitation and recurrence. (B) recurrent 4T1 tumor growth. (C) re-stimulating 4T1 tumor growth. (D) And (F) measuring central memory T cells (TCM, CD 62L) in blood at day 14 after DCHL treatment by flow cytometry ⁺ CD44 ⁺ ) With CD3 ⁺ CD8 ⁺ T cell ratio. (E) a growth curve of the re-stimulated tumor in each mouse. (G) TUNEL staining analysis of tumor tissue with recurrence and re-excitation of various treatment treatments. Scale bar: 20 μm. Data are expressed as mean ± SD, × P<0.01，***P<0.001; student t test.

Detailed Description

The following examples are provided to further illustrate the present invention and should not be construed as limiting the invention, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent substitutes.

Example 1 preparation and Performance identification of platelet exosome hybrid liposomes (DCHL) co-carrying DPDPD and CPO preparation of platelet exosome hybrid liposomes (DCHL) co-carrying DPD and CPO

(1) Synthesis of DPDPDPY

The synthetic route for DPDPy is as follows:

the method comprises the following specific steps: 4-dimethylaminocinnamaldehyde (0.22 g,1.28 mmol) and 1, 4-dimethylpyridine iodide (0.27 g,1.16 mmol) were dissolved in ethanol (10 mL). A few drops of piperidine were added and the reaction mixture was refluxed for 5h. Cooled to room temperature. The crude product was further purified by flash column chromatography on silica gel eluting with dichloromethane and methanol (DCM: methanol=20:1, v/v) to give DPDPy (0.16 g, yield: 40%). ¹ H NMR(400MHz,CDCl ₃ ,δin ppm):8.77(d,J＝6.6Hz,2H),7.75(d,J＝6.5Hz,2H),7.50(m,1H),7.47(d,J＝8.8Hz,2H),7.02(d,J＝15.4Hz,2H),6.83(m,3H),4.45(s,3H),3.05(s,6H).HRMS(MALDI-TOF(m/z):[M] ⁺ calcd for C ₁₈ H ₂₁ N ₂ ⁺ ,265.1699；found,265.1701.

DPDPy has AIE characteristics (fig. 2A and 2B) and good singlet oxygen generation capability (fig. 2C), as well as excellent photobleaching resistance (fig. 2D).

(2) Preparation of Platelet Exosomes (PEVs)

Platelet exosomes were prepared according to the method described in literature (Q.Ma, Q.Fan, J.Xu, J.Bai, X.Han, Z.Dong, X.Zhou, Z.Liu, Z.Gu, C.Wang, calming Cytokine Storm in Pneumonia by Targeted Delivery of TPCA-1Using Platelet-Derived Extracellular Vesicles, matter 2020,3,287-301.). The preparation method comprises the following steps: whole blood was collected from BALB/c mice sinuses, resuspended in PBS containing EDTA (5 mM, sigma-Aldrich) and PGE1 (1 mM, MCE), and then centrifuged at 100g for 15min to remove erythrocytes. Collect supernatant and centrifuge 8000g for 20min. The pellet was resuspended, activated with thrombin (2U/mL, solarbio) for 30min, and centrifuged at 800g for 10min. The collected supernatant was further ultracentrifuged at 100000rpm for 2 hours to obtain Platelet Exosomes (PEVs).

(3) Preparation of platelet exosome hybrid liposomes (DCHL) co-carrying DPDPDy and CPO

27mg of 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 2.8mg of cholesterol (Chol) and 3mg of DPDPDPPy were dissolved in chloroform and then evaporated in a rotary evaporator at 55℃for 80min to form a thin film. A PBS solution containing 4KU of chloroperoxidase (CPO, available from Macklin reagent) was then added to hydrate the film at 37℃for 5min, sonicated, then PEV (3 mg protein) was added, and repeated extrusion through a polycarbonate film having a pore size of 100 nm. The resulting DCHL particles were dialyzed overnight in dialysis bags (MWCO 300 KD) to remove unencapsulated DPDPPy and CPO. CPO and DPDPDPy loadings were calculated by UV-visible spectroscopy using a UV-visible spectrophotometer Lambda 35 (Perkin-Elmer). Load = M _drug /M _DCHL Wherein M refers to mass.

Blank platelet exosome hybrid liposomes (BL) were prepared in the same manner except that PBS was used instead of drug.

Liposomes (DCL) co-loaded with DPDPy and CPO were prepared in the same manner except for PEV removal.

DPDPDPy-loaded platelet exosome hybrid liposomes (DHL) were prepared in the same manner except CPO was removed.

2. Characterization of characteristics

TEM examination of the morphology of each liposome prepared, as shown in FIG. 3A, DCL showed a distinct liposome fingerprint structure with a relatively uniform size; platelet-derived exosomes have a dished structure, and DCHL has a morphology similar to liposomes. The hydrated particle size and zeta potential of DCL, PEV and DCHL were measured by DLS and the result is shown in fig. 3B and 3C, with zeta potential of DCHL being about-15.3 mV, lower than DCL (about-8.2 mV), probably due to the introduction of PEV membrane proteins. DCHL had good stability in PBS or FBS (fig. 3D).

By using

Resonance Energy Transfer (FRET) confirmed fusion of synthetic Liposomes (LIP) and PEV. The PEV membrane is labeled with a pair of FRET dyes, 1 '-octacosyl-3, 3' -tetramethylindocarbocyanine-4-chlorobenzenesulfonate (DiD) is the fluorescence donor, 1,1 '-octacosyl-3, 3' -tetramethylindole carbocyanine perchlorate (DiI) is the fluorescent acceptor, and thereafter is incrementally fused with LIP. In FIG. 3E, fluorescence recovered at 565nm with increasing LIP amount, but gradually decreased at 670nm, indicating that lipids intercalate into PEV membrane, resulting in reduced FRET interaction. CD41 and P-selectin proteins were detected in both PEV and DCHL (fig. 3F), indicating successful fusion of PEV and DCL. The uv-vis absorption spectrum of DCHL showed that absorption peaks for CPO and DPDPy occurred in DCHL, indicating that DPDPy and CPO were successfully loaded (fig. 3G). The drug delivery efficiencies (DLE) of DCHL on DPDPy and CPO were 82.3% and 34.7%, respectively, as measured by BCA protein quantification and uv absorption.

The singlet oxygen generation of DCHL under different conditions was analyzed by DPBF degradation experiments, and 0.05mL of different formulations were added to 2mL of DPBF: DCHL, PBS, DCHL and H ₂ O ₂ And NaCl (DCHL+H) ₂ O ₂ +NaCl), white light irradiated DCHL (DCHL+L), DCHL+L+H ₂ O ₂ +NaCl. The mixture was left in the dark for 30min using white light (0.1W/cm) ² ) As a light source, the absorption spectrum of the sample was measured at different times. As shown in fig. 3H and 3I, DCHL can cause significant DPBF degradation under light conditions. DCHL can also lead to significant degradation of DPBF in the presence of hydrogen peroxide and sodium chloride due to CPO catalysis. Therefore, DCHL can efficiently produce singlet oxygen, both in vivo and in vitro as a tumorTherapeutic potential nano-drug.

To study the stimulation of CPO release by laser irradiation, 10mL of DCHL containing 1KU CPO was added to the petri dish with or without 0.1W/cm ² CPO release experiments were performed with white light illumination (400-700 nm) for 3min and released CPO was determined using the bis-quinolinecarboxylic acid (BCA) protein assay. Under light irradiation, CPO can be released rapidly (FIG. 3J), which can be attributed to the destruction of the phospholipid bilayer by singlet oxygen generated by PDT.

4T1 cells were seeded in 24-well plates and cultured for 12 hours, and then 100. Mu.L of DiO-labeled DCL or DCHL (containing 0.01mg of DPDPDPy) was added to the medium. Then, the cells were incubated at 37℃with 5% CO ₂ Incubate for 1h and wash 3 times with PBS. Cells were harvested, stained with DAPI, imaged using a laser confocal microscope (CLSM), and fluorescence intensity detected by flow cytometry. As shown in fig. 3K and 3L, after 1h incubation, the fluorescence of DPDPy in tumor cells of DCHL group was significantly stronger than that of DCL group, which indicates that DCHL can adhere to tumor cells in a short time, which also provides basis for targeted treatment of DCHL under in vitro and in vivo conditions.

The result shows that the DCHL nanometer system can target tumor cells and has good singlet oxygen generation capacity.

Example 2 in vitro antitumor Capacity of DCHL

1. Experimental method

Cell line used in the experiments 4T1 mouse mammary cancer cell line was cultured in RPMI-1640 medium containing 10% FBS at 37℃with 5% CO ₂ Is cultured in a humidified incubator.

(1) Mitochondrial integrity assay

4T1 cells were seeded in 6-well plates and cultured overnight. Culturing for 2h after the following grouping treatment: (1) PBS; (2) L (0.1W/cm) ² 5 min); (3) dpdpy+l; (4) DHL; (5) DHL+L. The DPDPDPy concentration is 10. Mu.g/mL. After removal of the residual nanomaterial, a MitoTracker Red solution was added and incubated for 30min. After removal of the medium, the cells were washed three times with PBS and finally fluorescent images were recorded using CLSM.

(2) Intracellular H ₂ O ₂ Measurement

Detection of 4T1 thinCells were inoculated in 6-well plates and cultured overnight. Culturing for 2h after the following grouping treatment: (1) PBS; (2) L (0.1W/cm) ² 5 min); (3) dpdpy+l; (4) DHL; (5) DHL+L. The DPDPDPy concentration is 10. Mu.g/mL. Will be dissolved in RPMI-1640 (final concentration 50X 10) ^-6 M) H ₂ O ₂ Indicator (BES-H) ₂ O ₂ -Ac) was incubated with the cells for 40min. After removal of the medium, the cells were washed three times with PBS. Finally, the fluorescent image was recorded using CLSM.

(3) Intracellular ROS production

4T1 cells (1.5X10) ⁵ Well) were inoculated in 12-well plates and cultured for 12 hours. The packet processing is as follows: (1) PBS; (2) L (0.1W/cm) ² 5 min); (3) DCHL; (4) dcl+l; (5) dhl+l; (6) DCHL+L. The DPDPDPy concentration is 10. Mu.g/mL. After 0 or 4h of irradiation, 10×10 of the solution is dissolved ^-6 RPMI-1640 of M SOSG (singlet oxygen sensor green fluorescent probe, available from Dalian Biotechnology Co., ltd.) was incubated with cells for 10min, and then samples were observed under CLSM. The fluorescence intensity was measured by flow cytometry.

(4) DCHL induces immunogenic death of tumor cells (immunologic cell death, ICD)

4T1 cells were seeded in 48-well plates (2X 10 per well) ⁴ Individual cells) 12h later. The packet processing is as follows: (1) PBS; (2) L (0.1W/cm) ² 5 min); (3) DCHL; (4) dcl+l; (5) dhl+l; (6) DCHL+L. The DPDPDPy concentration is 10. Mu.g/mL. Cells were again cultured for 24h and 20. Mu.L of medium was used for detection in HMGB1 (high mobility group protein B1) ELISA (HMGB 1 ELISA kit was purchased from Solarbio technologies Co., ltd. In Beijing). Cells were then washed 3 times with PBS, fixed with 4% PFA, permeabilized with 0.1% Triton X-100 for 10min. After washing 3 times with PBS, cells were blocked with 10% FBS and washed with anti-CRT antibodies (Alexa

647 For 30min. Cells were washed 3 times with PBS and then stained with DAPI for 20min. Finally, cells were washed 3 times with PBS and observed using CLSM. ImageJ software measured fluorescence intensity.

(5) In vitro anticancer effect of DCHL

4T1 cells in six well plates at 37℃in 5% CO ₂ Incubating for 24 hours; thereafter, usingNew media replacement media cells were incubated in 6 different groups: (1) PBS; (2) L (0.1W/cm) ² 5 min); (3) DCHL; (4) dcl+l; (5) dhl+l; (6) DCHL+L. After a further 48h incubation, the cell activity was detected by means of the MTT cytotoxicity detection kit (Beyotime biotech.inc.).

2. Results

After successful preparation of DCHL, the mitochondrial damage capacity of DCHL on tumor cells was investigated, and the MitoTracker Red was used to investigate possible changes in mitochondrial membrane potential. As shown in fig. 4A, the red fluorescence of mitochondria was significant in PBS group, L group, DHL group, and barely red fluorescence in dhl+l group and dpdpy+l group, indicating that both mitochondria were damaged. As shown in fig. 4B and 4C, the dhl+l and dpdpy+l groups produced significant hydrogen peroxide. Subsequently, the introduction of CPO to study the continuous generation of singlet oxygen by DCHL in tumors, as shown in fig. 4D and 4E, DCL, DHL or DCHL can generate a large amount of singlet oxygen under light irradiation. However, after 4 hours, most cells of the DCHL-only group were observed to retain green fluorescence (SOSG). After 4h, by flow cytometry analysis, more than 70% of the cells in the dchl+l group maintained high levels of singlet oxygen, while the DHL and DCL groups had low levels of high levels of singlet oxygen in a large number of cells, with only a small fraction of the cells still possessing high levels of singlet oxygen. For the DCL+L group, because of the lack of targeting ability of the platelet exosomes, the concentration of CPO entering tumor cells is low, and EDT cannot be conducted efficiently, so that a large amount of singlet oxygen is generated. For the dhl+l group, it was not possible to maintain high levels of singlet oxygen for long periods of time due to lack of CPO-mediated EDT effects. Thus, DCHL can achieve uninterrupted singlet oxygen production by tumor cells.

Further, the growth inhibitory capacity and immunogenic death-inducing effect of DCHL on 4T1 cells were studied. As shown in fig. 5A, the tumor cell viability was lower than 10% for the dchl+l group, while the dhl+l group was close to 40%. This suggests that the introduction of CPO can significantly enhance the antitumor effect of DCHL. However, dcl+l group tumor cells showed higher survival, probably due to weaker targeting ability and insufficient intracellular internalization. DCHL was concentration-dependent on inhibition of tumor cell growth (fig. 5B). Singlet oxygen can react with intracellular GSH to deplete GSH, thereby promoting further oxidative damage to tumor cells. As shown in fig. 5C, the intracellular GSH content was the lowest in the dchl+l group, probably due to the sustained production of singlet oxygen by the dchl+l group. Intracellular GSH can participate in various physiological activities, plays an important role in tumor recurrence and drug resistance, and better GSH consumption capability enables DCHL to promote the curative effects of radiotherapy, chemotherapy and other therapies. The effect of DCHL on tumor immunogenic death was next studied. As shown in fig. 5D-5E, high mobility group box B1 (HMGB 1) is a highly conserved nucleoprotein, which induces Immunogenic Cell Death (ICD), enhances anti-tumor immunity, and exhibits remarkable anticancer effects. After DCHL+L treatment, the tumor cells release more HMGB1 protein and display obvious CRT fluorescence, which shows that the DCHL+L can induce the immunogenic death of the tumor cells, thereby providing a solid foundation for the subsequent T cell immunotherapy.

EXAMPLE 3 in vivo anti-tumor Capacity of DCHL

1. Experimental method

(1) The experimental animals were female BALB/c mice 5-6 weeks old, purchased from Vetong LiHua Corp (Beijing, china). Animal experiments were performed according to the protocol approved by the Ministry of health of the people's republic of China and approved by the animal research administration Commission of the people's hospitals in Shenzhen city.

(2) In vivo pharmacokinetic and distribution studies

Balb/c mice were subcutaneously injected 5X 10 on the right ventral side ⁶ 4T1 cells, when the tumor reached 300mm ³ At this time, balb/c mice (n=3) were intravenously injected with 100. Mu.L of DCL or DCHL in PBS (DPDPPy equivalent dose 10 mg/kg). At various time points after injection (i.e. 0.5, 1,2, 4, 8, 24 and 48 h), 20 μl of plasma was collected from the tail vein and then centrifuged at 10000rpm for 10min. Finally, the supernatant was collected and analyzed quantitatively for DPDPy concentration by fluorescence spectroscopy (FLS 980).

Balb/c mice were subcutaneously injected 5X 10 on the right ventral side ⁶ 4T1 cells, when the tumor reached 300mm ³ At this time, tumor-bearing mice (n=3) were injected intravenously with 100 μl of PBS containing DiR-labeled DCL or DCHL (DPDPy dose 10 mg/kg). Mice were imaged using the IVIS system 6, 12 and 24 hours after injection. Mice were then sacrificed 24h post injection, tumors and major organs were collected by using the IVIS systemLine imaging analysis and fluorescence intensity measurement.

(3) Evaluation of intratumoral oxidative stress

Balb/c mice were subcutaneously injected 5X 10 on the right ventral side ⁶ 4T1 cells, when the tumor reached 300mm ³ At this time, mice were randomly divided into 6 groups (3 per group): (1) PBS; (2) L (0.1W/cm) ² 5 min); (3) DCHL; (4) dcl+l; (5) dhl+l; (6) DCHL+L. The DPDPDpy dose was 10mg/kg. SOSG was injected intratumorally into tumor tissue before light exposure, and the tumor tissue was immediately removed after laser irradiation for frozen section staining, and frozen sections were observed by Confocal Laser Scanning Microscopy (CLSM).

(4) In vivo anti-tumor study

5X 10 injections were subcutaneously administered to the right ventral side of Balb/c mice, respectively ⁶ 4T1 cells (primary tumor) were subcutaneously injected 1X 10 to the left ventral side ⁶ 4T1 cells (distant tumor). Mice were first randomly divided into 6 groups (each group comprised of 5): (1) PBS; (2) L (0.1W/cm) ² 5 min); (3) DCHL; (4) dcl+l; (5) dhl+l; (6) DCHL+L. The DPDPDPy dose is 5mg/kg. All groups of mice were monitored for body weight and tumor volume every 3 days. Tumor length and tumor width were measured using calipers and tumor volume was calculated according to the following formula. Tumor volume = tumor length x tumor width ² /2. After 15d of treatment, mice were sacrificed. Blood samples from these mice were collected for biochemical analysis of blood. All mice were harvested for 5 major organs (heart, liver, spleen, lung and kidney), washed with PBS, and fixed with paraformaldehyde for histological analysis. And tumor tissue was weighed, 4% neutral buffered formalin fixed, paraffin treated conventionally, and 4 μm sectioned. Primary foci sections were then HE and CRT stained and finally examined using an optical microscope (BX 51, olympus, japan) and a fluorescence microscope (IX 81, olympus, japan).

(5) Investigation of the immune response in vivo after different treatments

To detect maturation of DCs in vivo, inguinal Lymph Nodes (LNs) were collected. Immunofluorescent staining with anti-CD 80-BV421 (BD Bioscience) and anti-CD 86-APC (ab 218757) antibodies was then followed by CD11c ⁺ Cell sorting kit (novo biotechnology co., ltd.) and flow cytometry to examine the rate of DC maturation in LN. To study T cell content in distant tumors, tumors were harvested from different groups of mice and mouse CD3 was used according to the manufacturer's protocol ⁺ T cell sorting kit (beaver biomedical engineering Co., ltd., suzhou), anti-CD 8-Alexa Fluor 488 (ab 237364) and anti-CD 4-PE (ab 252151) antibodies were treated. Remote tumor tissue was cut into small pieces and placed in a glass homogenizer with PBS (pH 7.4) containing 2% heat-inactivated fetal bovine serum. Then, a single cell suspension was prepared by gentle pressure using a homogenizer without adding digestive enzymes. Finally, CD3 was isolated ⁺ T cells and stained with a fluorescently labeled antibody after removal of the red blood cells using RBC lysis buffer. To analyze the cytokine secretion induced by the administration, whole blood of mice was collected 2 days after the administration. Serum concentrations of pro-inflammatory cytokines (including TNF- α and IFN- γ) were then analyzed with ELISA kits (Neobioscience co., ltd., china) according to the manufacturer's instructions.

(6) Studies for inhibition of tumor re-excitation and recurrence

To study the antitumor re-excitation and recurrence effects of DCHL, primary tumor-bearing mice received the various treatments described above. After excision of the primary tumor, the secondary 4T1 tumor was again stimulated in the left hind leg of the mouse. The growth of secondary tumors was recorded at regular intervals. Blood samples were collected on day 14 after the first dose for an immunological memory study. Use of mouse CD3 ⁺ And CD8 ⁺ T cell isolation kit (NovoBiotechnology Co., LTD.) for isolation of live CD3 ⁺ CD8 ⁺ T lymphocytes. The T cell subsets were finally analyzed on a flow cytometer by staining with antibodies (anti-CD 62L-BV421 and anti-CD 44-APC-Cy 7).

2. Results

(1) In vivo antitumor ability of DCHL

Compared to DCL, DCHL has better long-term circulating capacity and tumor targeting capacity (fig. 6A and 6B), showing the advantage of fusion liposomes in drug delivery. A bilateral breast tumor model was next established by subcutaneously injecting 4T1 cells into the left and right regions of the mice (fig. 7A). The left and right tumors are primary foci and distant tumors, respectively. After 7d inoculation, the mice were randomly divided into 6 groups (n=5 per group): (1) PBS; (2) L (0.1W/cm) ² 5 min); (3) injecting DCHL; (4) Post injection DCL illuminationShot (dcl+l); (5) light irradiation after injection of DHL (dhl+l); (6) light irradiation after DCHL injection (DCHL+L). Groups 4, 5, 6 were irradiated with white light only for 5min on the right tumor (primary focus). Tumor size (fig. 7B and 7C) and tumor weight were recorded every three days during treatment, see fig. 7D. After dchl+l treatment, primary foci of growth was significantly inhibited, as was distant tumors in dchl+l mice. The distant tumor growth curve of mice during treatment is shown in fig. 7E.

The therapeutic mechanism of dchl+l was further studied by detecting Dendritic Cells (DCs) in the lymph nodes, infiltration of T cells in distant tumors, and serum pro-inflammatory cytokines. DCs play a critical decision cell role in immune responses by participating in the initiation, regulation and maintenance of both innate and adaptive immune responses. T lymphocytes, which bind to co-stimulatory molecules (CD 80, CD 86), are important indicators of induction of T cell-mediated immune responses, markers of DC maturation. Lymph node dendritic cell maturation and CD4 in distant tumors in different treatment groups ⁺ /CD8 ⁺ The results of flow cytometry analysis of T lymphocytes are shown in fig. 7F. DCHL+L constituted a mature DC percentage of 50.3%, 1.47 times (34.3%) that of the DHL+L group, which was increased by about 4.07 times (9.93%) compared to the control group. And (3) measuring the concentration of cytokines in serum by adopting an enzyme-linked immunosorbent assay, and evaluating the systemic immune response induced by DC maturation. In contrast to the DCL+L and DHL+L groups, DCHL+L treatment consistently promoted secretion of IFN- γ and TNF- α (FIGS. 8A and 8B). CD8 in distant tumors ⁺ The number of T cells was significantly increased (FIG. 9), indicating that the combined use of DCHL and laser therapy gave a stronger anti-tumor immune response. In addition, SOSG was injected into tumors 12h after laser irradiation of the primary foci, and it was found that only tumor sections of dchl+l group showed strong SOSG and CRT fluorescence (fig. 7G). HE results showed significant damage to tumor tissue. These may be the cause of the better immune response in the dchl+l group. Throughout the dosing period, mice had no significant loss of body weight (fig. 10A), no significant impairment of liver and kidney function and tissue (fig. 10B and 10C), indicating good biosafety of DCHL.

(2) In vivo immune memory effect analysis

The immune memory effect is critical for durable tumor suppression and relapse prevention. As shown in fig. 11A, tumor recurrence was observed and a tumor re-challenge experiment was performed to reveal the anti-tumor immune memory effect of DCHL in primary 4T1 tumor-bearing mice. As shown in fig. 11B, all mice developed tumor recurrence. Whereas the dchl+l treated mice grew significantly slower than the other control groups. Next, mice receiving the different treatments were re-challenged with secondary homoneoplasms after surgical removal of the primary tumor. As shown in fig. 11C, the benefits of DCHL, dcl+l, and dhl+l treatment in inhibiting secondary tumors were negligible compared to PBS group. However, secondary tumor growth was inhibited in mice receiving dchl+l. These results indicate that dchl+l treatment elicits a sustained immune response against secondary tumors. The central memory T cells (central memory Tcells, TCM) survive longer in vivo, proliferate and differentiate into potent memory T cells upon stimulation with tumor antigens, thus playing a key role in long-term anti-tumor responses. Thus, the immune memory effect was studied by examining the proportion of TCM. As shown in fig. 11D and 11F, DCHL treatment caused a negligible increase in TCM ratio compared to PBS treatment. In contrast, dchl+l treatment resulted in a significant increase in TCM proportion. Furthermore, TUNEL fluorescence was observed in both dchl+l and dhl+l groups in tumor sections of recurrent and re-stimulated tumors, while other groups of tumors showed little apoptosis. These results indicate that treatment with DCHL in combination with PDT and EDT elicits a strong immune memory response.

Experimental results of a mouse subcutaneous bilateral tumor model, tumor recurrence and a re-excitation model show that DCHL treatment of primary foci can lead to substantial inhibition of distant tumors, the number of activated DC cells in the lymph nodes of the mice and CD8 in distant tumors ⁺ T cells are all significantly increased, causing a systemic immune response. After surgical excision, the number of central memory T cells in blood is obviously increased, and tumor re-excitation and recurrence can be obviously delayed. The DCHL provides a new strategy idea for tumor treatment.

Claims (10)

1. A delivery system for enhancing tumor immunotherapy, characterized by: the administration system for enhancing tumor immunotherapy is platelet exosome hybrid liposome loaded with AIE photosensitizer and chloroperoxidase.

2. The delivery system for enhancing tumor immunotherapy according to claim 1, wherein: the AIE photosensitizer is DPDPPy with a structure shown in the following formula:

3. the delivery system for enhancing tumor immunotherapy according to claim 2, wherein: the DPDPDPy is prepared by a method comprising the following steps: dissolving 4-dimethylamino cinnamaldehyde and 1, 4-dimethyl pyridine iodide in a solvent, adding piperidine, and carrying out reflux reaction to obtain DPDPDPy.

4. A delivery system for enhancing tumour immunotherapy according to claim 3, wherein: the solvent is ethanol.

5. A method of preparing a delivery system for enhancing tumour immunotherapy according to any of claims 1-4, characterized in that: the method comprises the following steps:

(1) Dissolving 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, cholesterol and AIE photosensitizer in an organic solvent, and forming a film by rotary evaporation; adding a solution containing chloroperoxidase to hydrate the film, and performing ultrasonic treatment to obtain a liposome carrying the AIE photosensitizer and the chloroperoxidase;

(2) Mixing the AIE photosensitizer and chloroperoxidase-loaded liposome and platelet exosome, and extruding to obtain the AIE photosensitizer and chloroperoxidase-loaded platelet exosome hybrid liposome.

6. The method of preparing a delivery system for enhancing tumor immunotherapy according to claim 5, wherein: in the step (1), the organic solvent is chloroform; the rotary steaming is carried out at 30-70 ℃; the hydration is carried out at 37 ℃; the solution containing the chloroperoxidase is PBS solution containing the chloroperoxidase.

7. The method of preparing a delivery system for enhancing tumor immunotherapy according to claim 5, wherein: in step (2), the extrusion is performed using a polycarbonate film having a pore size of 100 nm.

8. Use of the delivery system for enhancing tumour immunotherapy according to any of claims 1-4 for the preparation of an anti-tumour medicament.

9. An antitumor drug, characterized in that: a delivery system comprising the enhanced tumor immunotherapy of any of claims 1-4.

10. An antitumor drug according to claim 9, characterized in that: comprising a pharmaceutically acceptable carrier or excipient.

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